Bottom Line:
Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can "clamp" the ligand into a favorable binding pose.Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium.With the important role of structure-based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods.

Mentions:
The structure of the N1 calcium binding site includes close ion contacts with the backbone carbonyls of D293, N294, G297, G345, A346, Y347, and the carboxyl groups of both D293 and D324, with similar interactions observed in group 1 and 2 crystal structures.1–4, 9 Therefore, removal of the ion can be expected to destabilize these residues and the loops on which they are located, leading to increased fluctuation. Both GROMOS96 and AMBER FF99SB ion-bound MD simulations were checked for similar RMSF per residue Cα (Supporting Information Fig. 1), with the most flexibility observed for Loop 150, Loop 430, and portions of a long disordered loop (residues 327–348) surrounding the calcium binding site. Plots of the difference in ion-free and ion-bound simulation RMSF (Fig. .1) reveal changes in backbone RMSF for these regions. Both AMBER FF99SB and GROMOS96 simulations indicate increased fluctuation near the calcium-contacting residues (see peaks near asterisks in Fig. .1), with some of the peaks corresponding to residues very close to the active site. RMSF changes are color-mapped onto the N1 monomer structure in Supporting Information Figure 2, for an overall view of where these changes occur in the protein. Loop 150 and 430 (labeled in Fig. .1) have changes in flexibility that are inconsistent in comparison of the GROMOS96 and AMBER FF99SB simulations.

Mentions:
The structure of the N1 calcium binding site includes close ion contacts with the backbone carbonyls of D293, N294, G297, G345, A346, Y347, and the carboxyl groups of both D293 and D324, with similar interactions observed in group 1 and 2 crystal structures.1–4, 9 Therefore, removal of the ion can be expected to destabilize these residues and the loops on which they are located, leading to increased fluctuation. Both GROMOS96 and AMBER FF99SB ion-bound MD simulations were checked for similar RMSF per residue Cα (Supporting Information Fig. 1), with the most flexibility observed for Loop 150, Loop 430, and portions of a long disordered loop (residues 327–348) surrounding the calcium binding site. Plots of the difference in ion-free and ion-bound simulation RMSF (Fig. .1) reveal changes in backbone RMSF for these regions. Both AMBER FF99SB and GROMOS96 simulations indicate increased fluctuation near the calcium-contacting residues (see peaks near asterisks in Fig. .1), with some of the peaks corresponding to residues very close to the active site. RMSF changes are color-mapped onto the N1 monomer structure in Supporting Information Figure 2, for an overall view of where these changes occur in the protein. Loop 150 and 430 (labeled in Fig. .1) have changes in flexibility that are inconsistent in comparison of the GROMOS96 and AMBER FF99SB simulations.

Bottom Line:
Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can "clamp" the ligand into a favorable binding pose.Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium.With the important role of structure-based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods.